identification of genes involved in the regulation of
TRANSCRIPT
Identification of genes involved in the regulation of sensory organ precursor formation in Drosophila melanogaster
Research Thesis
Presented in partial fulfillment of the requirements for graduation with research distinction in Molecular Genetics in the undergraduate college of The Ohio State University
by
Jared Kusar
The Ohio State University June 2012
Project Advisor: Professor Harald Vaessin, Department of Molecular Genetics
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Acknowledgements
I am grateful for Harald Vaessin in providing the opportunity to learn and experience
what scientific research is. His guidance has helped to shape myself as a beginning scientist, and
on this project. Yifei Zhang helped in collaboration on our work with cul-2. Kristen Bremer was
instrumental with developing my dissection techniques, and Maki Asano for her RNAi lines.
Megan Weidrick aided in fixing grammatical issues. Support from the Undergraduate Research
Scholarship made this project possible. Most of all I want to thank my parents Jerry and Pam
being there every step of the way in my life.
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Abstract
Defects in early neurogenic development are associated with a wide range of pathological
conditions. The Fred protein, a transmembrane IgC2 protein, is necessary for normal
development and critical for the Notch signaling pathway. Using Drosophila melanogaster as the
model, the regulatory roles of other genes can be studied that exhibit functional interactions with
these genes. Macro- and micro-chaeta sensory bristles (large and small bristles) cover the adult
fly and provide an avenue to study cell fate. As each bristle originates from an individual sensory
organ precursor cell, the presence or absence of sensory bristles indicates sensory organ
precursor cell differentiation. From an RNAi mini screen of an array of candidate genes, 2 genes,
cullin-2 (cul-2) and tumbleweed (tum), were identified and exhibited a phenotype similar to that
of fred, specifically a loss of function. cul-2 is a scaffold protein for ubiquitin ligase, while tum is
involved in GTPase inactivation. An additional aim of this study is to understand how these two
genes interact within the fred pathway. The two genes were identified using the inducible RNAi /
GAL4/ UAS system. The GAL4/UAS system allows one to induce gene specific RNAi in
specific tissues and at defined developmental times. Observing the phenotypical consequences of
this down regulation occurs at two levels: (1) the adult animal, and (2) in imaginal wing disc of
late 3rd instar larvae. Additional studies have been started to further define the phenotype of
these genes and their respective interactions with fred gene function. Initial experimental
observations suggest that tum, indeed, may have additional roles in the regulation of muscle
attachment sites. Furthermore, double mutations of cul-2 and tum, tum and fred, tum and sc are
being analyzed to determine potential epistatic relationships.
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TABLE OF CONTENTS
I. Introduction……………………………………………………………………………...5 Background……………………………………………………………………………….5 What is cullin-2?..................................................................................................................5 What is tumbleweed?...........................................................................................................6 DNA microarray…………………………….…………………………………………….6
II. Approach/ Material and Methods………………………………………………………9
Stocks used………………………………………………………………………………..9 Fly chromosomes and balancers…………………………………………………………..9 GAL4/UAS system……………………………………………………………………….9 X-gal staining for wing discs…………………………………………………………….10 Digoxygenin-labeled RNAi probe………………………………………….……………10 cul-2 in-situ hybridization………………………………………………………………..11 Setting up double mutation crosses………………………………………………………12
III. Results…………………………………………………………………………………...17
Identification of fred interacting genes…………………………………………………..17 cul-2 adult phenotypes knockdown with pnr-GAL4, en-GAL4, ap-GAL4, and c765-GAL4 drivers……………………………………………………………………………………17 cul-2 knock-down induces ectopic SOPs outside proneural clusters……………………18 cul-2 in-situ………………………………………………………………………………18 tum adult phenotypes knockdown with drivers………………………………………….18 tum wing disc phenotype resembles cul-2 more than fred………………………………18 tum GAL80 temperature shift (ts) study………………………………………………...19 tum mosaic study………………………………………………………………………...20 Double mutations………………………………………………………………………...20
IV. Discussion.........................................................................................................................30 cul-2 is required for sensory organ precursors for suppression of sensory organ formation...........................................................................................................................30 tum is required for sensory organ precursors for suppression of sensory organ formation………………………………………………………………………………...31 tum has a later function in development…………………………………………………31 tum has a function in bristle formation…………………………………………………..32 tum in fred, tum in cul-2, and tum in sc interaction ……………...……………………...33
V. Conclusion………………………………………………………………………………35
VI. References………………………………………………………………………………36
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FIGURES
1. Prepattern hypothesis………………………………………………………………8
2. UAS-CG-RNAi stocks ……………………………………………………………...14
3. Fly crosses/ balancers………………………………………………………………15
4. GAL4/UAS-RNAi system………………………………………………………….16
5. Identification of fred interacting genes …………………………………………..21
6. cul-2 knockdown associated phenotypes in adult animal………………………..22
7. cul-2 knockdown associated phenotypes in wing disc……………………………23
8. cul-2 in-situ………………………………………………………………………….24
9. tum adult phenotypes………………………………………………………………25
10. tum suppression in 3rd instar wing disc results in ectopic SOP formation……...26
11. tum GAL80 temperature shift study driver points to possible late function of tum
…………………….……………………………………………………………..….27
12. tum GAL80 temperature shift study c765-GAL4 with driver…………………...28
13. tum mosaic study…………………………………………………………………...29
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Introduction
Background
The adult fly is covered in macrochaeta and microchaeta (large and small bristles), which
are sensory organs that help the animal sense its environment. Sensory organs arise from
(sensory organ precursors) SOP cells in the third instar larve. Three groups of genes regulate
sensory organ precursor formation according to the prepattern hypothesis: prepattern, proneural
and neurogenic genes. Prepattern genes mark overlapping regions of gene expression (Culi et al.,
1998). Proneural genes are expressed in so-called proneural clusters and result from prepattern
gene function (Campos-Ortega, 1993). Subsequently, proneural genes interact with Notch
signaling to limit neurogenic potential to a single SOP in each proneural cluster (Figure 1).
Previous work in the lab has identified a gene friend of echinoid (fred) (Chandra et al.,
2003), which is similar in sequence to echinoid (Ahmed et al., 2003). Echinoid is an
immunoglobulin C2-type cell-adhesion molecule (IgC2), which is involved with the Delta ligand
in the Notch pathway (Rawlings et al., 2003). Preliminary investigation in the Vaessin lab
suggests that there is an earlier regulating step prior to the prepattern genes. This earlier process
involves a requirement of fred function (Chandra). Subsequent work in the lab was enacted
towards the identification of fred interaction or downstream genes. The purpose of my study is to
better understand which genes, from a collection of genes that were found to be miss-expressed
early on in neurogenesis in a fred microarray study, function.
What is cullin-2?
cul-2 is located on the second chromosome and is a member of the cullin gene family that
plays a critical role in the ubiquitination pathway. Cullin 1-7 are scaffolding proteins that make
up the cullin-RING E3 ubiquitin ligase (CRL). The ubiquitin-proteasome system works by
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attaching ubiquitin to an E1 activation enzyme in an ATP dependent reaction. This ubiquitin
subsequently is transferred to an E2 conjugation enzyme. The E3 ligase binds both substrate and
E2 which results in mono or polyubiquitination that will signal whether to be degraded by the
26S proteosome (Sarikas, 2011). Ubiquitination of proteins is essential for many protein
signaling processes some of which include tumor suppression, cell growth and signal
transduction. There are six cullin genes in the Drosophila genome (Hudson et al., 2010), and
eight members in mammals. cul-2 function is currently not well understood in mouse or
Drosophila melanogaster. In C. elegans cul-2 is involved in a range of physiological functions.
What is tummbleweed?
tum also known as RacGAP50C is located on the second chromosome. tum mutations
disrupt GTPase Activating Protein (GAP) (Goldstein et al., 2005). GAP and guanine nucleotide
exchange factors (GEF) work as genetic switches in the cell to relay signals by either activating
or deactivating GTPase (Somers et al., 2003). The ras gene, which is a monomer GTPase, is
mutated in 1 in 5 of all human cancers (Alberts et al., 2008). It is proposed that tum serves as a
scaffolding protein (Goldstein). Another known function of RacGAP50C is that it regulates
cytoskeleton required for cytokininesis, and is required to limit axon growth (Goldstein 2005). It
connects the contractile ring to cortical microtubules at the site of furrowing in dividing cells and
negatively regulates the wingless pathway during Drosophila embryonic development, and is
required for neuroblast proliferation and limits axon growth (Goldstein).
DNA microarray
DNA microarray technology enables the study of genome wide patterns of gene
expression. The Vaessin lab had previously completed a microarray for fred-RNAi mRNA and
found 62 downregulated genes and 72 upregulated genes (unpublished results). Furthermore, an
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RNAi mini screen was performed on microarray confident genes and from a fred yeast two
hybrid screen. RNAi lines, where available, have been obtained for these genes and used for
phenotype observations.
Fifteen genes were initially studied by creating fly crosses and observing phenotypes at
the adult and wing disc level. cul-2 and tum were two genes identified to display adult
phenotypes in line with possible role in early neurogenesis, and also displaying ectopic (outside
of normal expression) SOP formation in imaginal wing discs. In this thesis I present the
identification of cul-2 and tum and evidence that cul-2 and tum are two new novel genes in early
neurogenesis.
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Figure 1: Prepattern hypothesis
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Approach/ Material and Methods
Stocks used
Fifteen UAS-CG-RNAi lines from Dr. Maki Asano and Vienna fly center were used for
initial identification crosses (Figure 2). Other stocks used: pnr-GAL4, ap-GAL4, c765-GAL4, en-
GAL4, neurA101-lacZ, hsFLP; FRT42tum347, FRT42GFP, tubP-GAL80ts. UAS-fred RNAi, sca-
lacZ.
Fly chromosomes and balancers
Flies have 4 pairs of chromosomes an X/Y pair and three autosomal chromosomes. Since
a fly is diploid and inherits one set of chromosomes from each parent, a tracking system is
needed. Balancers are used for this purpose. To accomplish this, balancer chromosomes
generally have: (1) multiple chromosome aberrations such as inversions and translocations, (2) at
least one dominant marker gene, and (3) frequently one recessive lethal mutation (Lindsley and
Grell, 1972). Using balancers allows one to visualize the genotype by looking at the phenotype
(Ashburner, 1989). An example of a balancer is Curly derivative of Oster (CyO). If a fly inherits
this marker it will have curly wing as a dominant tracking phenotype. Another balancer called
Stubble (Sb) results in a fly with shorter sensory bristles. Figure 3 depicts a hypothetical fly cross
and resulting progeny to demonstrate this point.
GAL4/UAS system
The GAL4/UAS system is a borrowed genetic system from yeast. GAL4 is a yeast gene
that encodes for the GAL4 protein, which is a transcription factor. Upstream Activation sequence
(UAS) is a GAL4 binding site on DNA only found in yeast that is the target of GAL4. Both
GAL4 and UAS by themselves do not do anything, but in combination GAL4 can bind UAS and
drive expression of coding sequence downstream of the UAS sequence. In flies we accomplish
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this by transgenic means. One fly stock in which the GAL4 gene has been introduced is crossed
with a UAS fly stock. A proportion of the offspring will have both GAL4 and UAS.
Furthermore, GAL4 lines are used that express GAL4 in a specific spatial/temporal
developmental context. The UAS sequence is part of a construct that also contains a gene of
interest to be miss-expressed (Figure 4). If this gene of sequence is an RNAi construct, via a
Palindrome hairpin sequence the target gene will be down regulated (Brand and Perrimon, 1993).
X-gal staining for wing discs
β-galactosidase activity can be detected by X-gal. This method was used on all fifteen
CG lines. en-GAL4>> UAS-CG-RNAi and pnr-GAL4;A101>>UAS-CG-RNAi. The resulting
flies are collected under CO2 and stored in 96% ethanol. Pictures are compiled using a dissection
microscope. Additionally, third instar larvae are collected for X-gal staining. Larvae are
dissected in ice cold PBS for no more than 10 minutes. Fixation of larvae in 1% glutaraldehyde
is done for 10 minutes. Samples are washed three times with PBT (PBS with 1% tween-20) 3
minutes each. The solution is removed and 10 ul of 10% X-gal solution in 490 ul X-gal staining
buffer is added. X-gal staining buffer is composed of 160 mg of Potassium Ferricyanide
Crystalline (5mM), 210 mg of Potassium Ferricyanide Trihydrate (5mM), 20 mg of Magnesium
Chloride (2mM), 100 ml PBS, and 100 ul of Tween-20. The reaction proceeds at room
temperature overnight. Reaction is stopped by washing 3 times for 3 minutes each with PBT.
Finally, discs are isolated and mounted in aquamount.
Digoxygenin-labeled RNA probe
Protocol for synthesizing DIG-labeling transcription reaction. Primers for cul-2 were
combined with genomic DNA. The antisense primer was constructed with a T7 promoter. Each
10 ul reaction should contain: DNA 3.5 ul, mMATP 0.75 ul, 0.75ul mMGTP, 0.75ul mMCTP,
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0.50ul mMUTP, 10x transcription buffer, 1.3 ul digoxygenin UTP, Roche MEGAscript T7 RNA
polymerase. Set at 37°C for 6 hrs and then stored overnight at 4°C.
cul-2 in-situ hybridization
In-situ hybridization allows one to visually locate the messenger RNA in the tissue. This
three-day process was used for cul-2. Tissue can be dehydrated in alcohol in a series of steps
(25%, 50%, 75% 96% Et-OH) after the first fixation and stored at -20°C on the first day.
Day 1: Dissect third instar larvae and invert in ice cold PBT in less than 10 minutes. Fixate the
tissue with 4% formaldehyde for 20 minutes. Wash in PBT 3 min each, 4 times.
Exchange solution for 500 ul of PBT with 2 ul of proteinase K. Quick wash 4 times in
PBT to stop the reaction by inverting after each exchange. Post fixation with 4%
formaldehyde 20 minutes followed with washing 4 times 3 minutes each in PBT.
Exchange into hybridization solution for 5 minutes, twice. Exchange again and incubate
for an hour at 55°C. Remove and add 1 ul of RNA probe to 50 ul of hybridization
solution. Heat to 95°C for 3 minutes, cool on ice for 3 minutes and add wing disc at
55°C overnight.
Day 2: Wash for 20 minutes each in hybridization solution at 55°C, 4 times. Washed 15 minutes
each at 50% and 25% hybridiazation solution at room temperature. Washed in PBT for 7
minutes each, 5 times. Meantime rehydrate embryos (25%, 50%, 75%,100% PBT), 2
minutes each step. Block 10 minutes with PBT and 1% BSA. Add antibody AntiDig
(1:500). Incubate for 1 hour at room temperature. Use this pre-incubated antibody and
add to the wing discs at a final (1:4000) dilution over night at 4°C.
Day 3: Wash for 15 minutes each in PBT, 4 times; followed by AP buffer wash, 2 times.
Remove last of AP buffer and add AP buffer with 4.5 ul NBT and 3.5 ul BCIP and place
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in viewing dish in dark. Check the color reaction frequently. Stop reaction with a drop of
ethanol. Wash with PBT and mount in aquamount.
Setting up Double mutation crosses
Double mutation crosses were performed with tum in cul-2, tum in sc, and tum in fred.
Below are the crosses for the resulting genetics. Step four in the series of crosses below was
established to create a stock.
UAS-tum-RNAi(II) in UAS-cul-20RNAi(III) double mutation crosses .
1) ♂ UAS-tum-RNAi/UAS-tum-RNAi x ♀ L/CyO; Sb/tb
2) ♂ UAS-tum-RNAi/CyO; +/sb x ♀ L/CyO; UAS-cul-2-RNAi/Tb
3) ♂ UAS-tum-RNAi/L;cul-2RNAi/Sb x ♀ Sb/CyO-tb
4) ♂ UAS-tum-RNAi; UAS-cul-2-RNAi/CyO-tb x ♀ en-GAL4; A101/CyO-tb
5) UAS-tumRNAi; UAS-cul-2RNAi/ en-GAL4; A101
UAS-tum-RNAi(II) in UAS-fred-RNAi(III) double mutation crosses
1) ♂UAS-tum-RNAi/UAS-tum-RNAi x ♀ L/CyO; Sb/tb
2) ♂UAS-tum-RNAi/L; +/tb x ♀ Sco/Cyo; UR3-3/Sb
3) ♂ UAS-tum-RNAi/Cyo; UR3-3/Tb x ♀Sb/CyO-Tb
4) ♂ UAS-tum-RNAi: UR3-3/ CyO-Tb x ♀ en-GAL4: A101/CyO-Tb
5) UAS-tum-RNAi: UR3-3/ en-GAL4:A101
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UAS-tum-RNAi(II) in UAS-sc-RNAi(III) double mutation crosses
1) ♂UAS-tum-RNAi/UAS-tum-RNAi x ♀L/CyO; Sb/Tb
2) ♂UAS-tum-RNAi/Cyo; +/Sb x♀ L/Cyo; Sc-RNAi/Tb
3) ♂UAS-tum-RNAi/Cyo; UAS-Sc-RNAi/ Sb x ♀ Sb/ Cy-Tb
4) ♂UAS-tum-RNAi; UAS-Sc-RNAi/CyO-Tb x ♀en-GAL4: A101/CyO-Tb
5) UAS-tum-RNAi; UAS-Sc-RNAi/ en-GAL4: A101
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Stock #/CG Name
1. 104160/1034 Bicoid
2. 105101/1512 cullin-2
3. 101538/2621 Shaggy
4. 100165/2909 unknown
5. 105562/3244 C-type lectin 27kD
6. 104523/4005 Yorkie
7. 101829*/4373 Cyo6d2
8. 100906/5210 chitinase-like
9. 100281/9390 Acetyl Coenzyme A synthase
10. 107130/10079 Epidermal growth factor receptor
11. 110392/10955 Rtf1
12. 101166/12283 kekkon-1
13. 106850/13345 Tumbleweed
14. 104496/17870 14-3-3
15. 101275/34395 Nubbin
Figure 2: UAS-CG-RNAi stocks used
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Figure 3: Fly crosses and balancers
In this hypothetical cross one fly with the dominant balancer Curly is crossed to a fly with the Stubble mutation. Four possible progeny are apparent and can be distinguished by the flies’ phenotype.
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Figure 4: GAL4/UAS-RNAi system A fly crossed to create progeny with both GAL4 and UAS will allow the UAS construct Gene X to be miss-expressed by the enhancer trap GAL4. The enhancer trap is expressed in a spatial/ temporal background. The downstream DNA coding sequence will hairpin if the order is a Palindrome.
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Results
Identification of fred interacting genes
In order to identify genes that potentially interact with fred, I used the small wing
phenotype of UAS-fits. The fred intercellular transmembrane domain sequence (fits) is construct
containing parts of the fred protein, specifically the intercellular domains and the transmembrane
domain. It was hypothesized that the genes which interact with fred may modify this phenotype.
Two types of crosses were used to misexpress CG-RNAi. First, I used ap-GAL4 misexpression to
drive UAS- fits with UAS-CG-RNAi (Figure 5). apterous (Ap) is expression in a large part of the
wing compartment and notum. It was observed that the fits animals show smaller wing
phenotypes, and the ap-GAL4 >>UAS-cul-2-RNAi animals have smaller wings and loss of
macrochaeta. The second cross c765-GAL4 was used to miss-express UR3-1 with UAS-CG-RNAi
(Figure 5). c765-GAL4 is expressed in the wing.
Smaller wings in c765-GAL4 >>UAS-cul-2-RNAi were observed. These identification
crosses identified another gene tum from the c765-GAL4 >>UR3-1;UAS-tum-RNAi cross (Figure
5). In this animal the wing marginal shows an increase in the number of bristles.
Cul-2 adult phenotype knockdown with drivers pnr-GAL4, en-GAL4, ap-GAL4, and c765-
GAL4
pannier-GAL4 (pnr-GAL4), engrailed-GAL4 (en-GAL4), apteros-GAL4 (ap-GAL4), and
c765-GAL4 were used to misexpress cul-2-RNAi in adult animals. pnr-GAL4 is expressed in the
median notum. en-GAL4 is expressed in the posterior compartment of the wing. ap-GAL4 shows
large expression in most of the wing and notum. c765-GAL4 is limited to the wing. I observed
missing macro and microchaetae in pnr-GAL4;A101>>UAS-cul-2-RNAi (Figure 6). The animals
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with the en-GAL4>>UAS-cul-2-RNAi and c765-GAL4>>UAS-cul-2-RNAi showed smaller
wings. Ap-GAL4 show smaller wings and loss of bristles on the notum.
cul-2 knock-down induces ectopic SOPs outside proneural clusters
Wing disc phenotypes using A101 staining with en-GAL4 and pnr-GAL4 drivers. en-GAL4
is expressed in the posterior region of the wing disc, while pnr-GAL4 is in the distal region. en-
GAL4>>UAS-cul-2-RNAi animals showed A101 expression indicating ectopic SOP cells. In the
control no ectopic SOP were observed (Figure 7). pnr-GAL4>>UAS-cul-2-RNAi also showed
ectopic SOP cells. These pheneotypes resember the fred-RNAi phenotype.
cul-2 in-situ
fred-RNAi knock down compared to cul-2-RNAi knock down displays stronger
phenotypes in the notum. To determine the gene expression of the cul-2 gene, in-situ
hybridization was performed. In the first instar larvae, general uniform expression in wing discs
was found. In the second day third instar larvae, the wing discs show less uniform expression
with higher levels of expression in the wing margin. cul-2 mRNA (Figure 8) shows that lower
levels in some regions of the wing disc would support the observation of weaker phenotypes
seen in the notum of the animals
tum adult phenotypes knockdown with drivers
In order to determine the adult phenotype with RNAi, tum-RNAi lines were crossed with
en-GAL4 and pnr-GAL4. In the progeny of these crosses, smaller thorax, smaller wings and loss
of macro and microchaetae existed (Figure 9).
tum wing disc phenotype resembles cul-2 more than fred
Using A101 neurogenic marker in the wing disc for SOP cells the disc phenotype was
shown for tum (Figure 10). The genetics were en-GAl4>>UAS- tum-RNAi and pnr-GAl4>>
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UAS-tum-RNAi show phenotypes that closely resemble the cul-2-RNAi wing disc phenotypes.
Even more so than the fred-RNAi wing disc phenotype.
tum GAL80 temperature shift (ts) study
tum is shown to have an early function in development. Any potentially later function of
tumbleweed would be masked by earlier events when using the GAL4/UAS-RNAi technique,
and has not yet been studied. GAL80ts (mutation of normal GAL80) is not functional as 29°C
degrees and is unstable (McGuire et, al., 2003). Here the GAL80ts/GAL4/UAS technique was
used to study tum interaction at different stages of development. The mechanism works as such:
GAL80ts encodes a protein in the nucleus that binds to the GAL4 protein. This inhibits the
transcription machinery to be recruited. At the restrictive temperature GAL80ts loses its intrinsic
function and no longer works (McGuire). In the experiment flies were created with the genotypes
of either GAL80ts-׀pnr-GAL4>>UAS-tum-RNAi (Figure 11) or GAL80ts -׀c765-GAL4>>-UAS-
tum-RNAi (Figure 12) and raised at 19°C through early stages in development. The flies were
then transferred to 29°C temperature setting where GAL80ts no longer inhibits GAL4, and thus,
GAL4 drives UAS-tum-RNAi expression at a later time in development. This enables one to test
for functions of tum at later stages of development.
“Regular suppression” with GAL80ts driven by c765GAL4 shows bigger wings with
thicker bristles in the wing marginal and different positioning of the wings compared to
“unmodified suppression” with no GAL80ts. “Regular suppression” with GAL80ts driven by
pnr-GAL4 on the other hand showed bulges in the epidermis. See “tum has a later function in
development” for discussion.
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tum mosaic study
FRT/FLP system: heat shock is used to induce flip protein, not by UAS, but with the
Hsp70 promoter. Heat shock induces Flip protein expression (Theodosiou and Xu, 1998). A
mosaic is a mixture of cells that have different genotypes within an organism. In this study
transgenic flies with the yeast recombinase flip gene is combined with its target FRT to create
mosaic flies. Homologous chromosomes undergo mitotic recombination when the animals are
heat shocked and FLIP is expressed. The animals were heat shocked in a water bath each day for
45 minutes at 38°C to induce recombination. Due to time limitations studies could not be
performed in the wing disc because all crosses must be created to have each animal with the
correct, corresponding, markers. The following genetics was used to create tum clone: hsFLP;
FRT42tum347/cy-tb x FRT;GFP/Cy. In this pilot experiment, I observed animals that had
multiple bristles from one socket, in addition to marginal wing bristles and mild rough eye
phenotype (Figure 13).
Double mutations
Formation of SOPs requires a function of the proneural scute (sc) gene. In order to
determine if scute can suppress or reduce the tum phenotype, I created a series of crosses with
UAS-tum-RNAi and UAS-cul-2-RNAi taking advantage of fly balancers, figure not shown. My
hypothesis is that sc can reduce ectopic SOP in the wing disc because sc is necessary for SOP
development. Furthermore, en-GAL4 was used to for misexpression. The second double mutation
tum in cul-2 was spawned to gain insight into any possible interaction between cul-2 and tum.
The last combination of double mutations created was tum and fred. The purpose of these crosses
was to determine the potential epistatic relationship of these genes. I observed ectopic SOP in the
wing discs and smaller wing. See discussion for an depth analysis.
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Figure 5: Identification of fred interacting genes
Animal (A) has the genetics of ap-GAL4>>UAS-fits; UAS-cul-2-RNAi. Notice the smaller wings and loss of macrochaetae compared to the control (B) ap-GAL4 >>UAS-fits. Animal (C) has the genetics c765-GAL4>>UR3-1; UAS-cul-2-RNAi and control (D) animal has c765-GAL4>>UR3-1 genetics. Experimental animal (C) has smaller wings than control (D). The animal in (E) has the genetics of c765-GAL4>>UR3-1; UAS-tum-RNAi and control (F) has c765-GAL4 >>UR3-1-fits. The control in (F) has larger wings than the experimental animal (E).
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Figure 6: cul-2 knockdown associated phenotypes in adult animal
cul-2 knockdown leads to loss of microchaeta and macrochaeta in (A) pnr-GAL4>>UAS-cul-2-RNAi. Control animal (B) is without the UAS construct. Notice smaller wings with other drivers in (C,E, G). Animal (C) has driver en-GAL4>>UAS-cul-2-RNAi. Control (D) animal no UAS. Animal (E) has driver ap-GAL4>>UAS-cul-2-RNAi. Animal (F) is a control with no UAS construct. Animal in (G) has c765-GAL4>>UAS-cul-2-RNAi. Animal (H) is a control with no UAS construct.
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.
Figure 7: cul-2 knockdown associated phenotypes in wing disc
neur-lacZ staining of wing discs with genotype (A) en-GAL4>>UAS-Cul-2RNAi. The wing disc (B) has genetics of pnr-GAL4>>UAS-Cul-2RNAi. The control in (C) has neur-lacZ. Notice in (A) and (B) ectopic SOPs compared to control (C).
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Figure 8: cul-2 in-situ
In 1st day third instar larva (A) we find general uniform expression in the wing disc compared to second day third instar larva (B) where the wing disc shows lower general expression with higher expression in the wing margin.
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Figure 9: tum adult phenotypes Suppression of tum in dorsal-central region with genotype pnr-GAL4>>UAS-tum-RNAi (A) displays macrochaeta and loss of epidermis. Wild type fly in (B and H). Flies with genotype en-GAL4>>UAS-tum-RNAi are depicted in (C-J excluding H). Notice the much stronger phenotypes in notum (C and D) compared to (F) and (I) animals.
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Figure 10: tum suppression in 3rd instar wing disc results in ectopic SOP formation
Ectopic SOPs evidence is shown in (A) wing disc with en-GAL4>>UAS- tum-RNAi genetics, and (B) pnr-GAL4 >>UAS-tum-RNAi genetics. Control wing disc (C) is shown for comparison. neur-lacZ staining was used in all cases above.
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Figure 11: tum GAL80ts temperature shift study driver points to possible late function of tum Animal in (A,C,E) have the genetics of GAL80ts-׀pnr-GAL4>>-UAS-tum-RNAi. Animal in (B,D, F) have the genetics of pnr-GAL4>>UAS-tum-RNAi . The day shifted matches horizontally with the graphs and descend with possible later functions of tum. GAL80ts animals show polarity differences, and less bristles than the no GAL80ts animals. Fly (G) depicts one later function of tum, bulges at the notum.
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Figure 12: tum GAL80 temperature shift study driver c765-GAL4
Animal with GAL80ts (A-C) show 19°C to 29°C shift and partial upright wings. Wild type (WT) is the normal wing placement. Image (D) is a close up of (C). Later function of tum in wing marginal (D) is shown. Notice smaller wings, double bristle and socket in (E) of animal without GAL80ts .
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Figure 13: tum mosaic study tum clone: hsFLP; FRT42tum347/FRT42-GFP. Animal (A) shows cluster of bristles from a single socket. Animal (B) also shows a small clone. Animal (C, E, G) are wild type for comparison. Animal (D) is experimental wing. Image (F) is a close up of (D). Notice wing marginal phenotype in (F).
30
Discussion
cul-2 is required for sensory organ precursors for suppression of sensory organ formation
cul-2 encodes the scaffolding protein involved in ubiquitination. A total loss of cul-2
results in non-development. Therefore cul-2 must be knocked-down in certain parts of the fly at a
time to be studied and then collectively compared. The purpose of this analysis was to determine
and identify genes that might be involved in the fred pathway. To this end a set of fifteen lines
were analyzed at the adult and third instar larva level. A total of two genes tum and cul-2 were
identified, both of which show qualitative similar phenotypes as fred. fits is a partial fred
construct that contains only fred intercellular domain and transmembrane region. Expression of
fits alone with ap-GAL4 results in smaller wing phenotype. I used the sensitive fred background
to see whether the RNAi mediated repression of candidate genes would modify this phenotype.
In following crosses with pnr-GAL4, en-GAL4, c765-GAL4, ap-GAL4 drivers of different
wing and/or notum expression, we have determined the phenotypes associated with cul-2
knockdown. Specifically, I observed smaller wings, loss of macrochaeta, and a reduction of
epidermis. These observations by themselves do not necessarily show that a cul-2 knockdown is
associated with SOP formation. To test this the SOP marker, neur-lacZ, which is specifically
expressed in SOP cells, was used. en-GAL4>>UAS-cul-2-RNAi and pnr-GAL4>>cul-2-RNAi
(figure 7) show ectopic A101 expression. These results mean that cul-2 is required to suppress
SOP formation in Drosophila wing disc cells. Additional genetic interaction studies involving
Notch and cul-2 draw conclusions that Notch signaling is not activated upon knock-down of cul-
2. Experimental data for the ligand of Notch, Delta further supports this by being up regulated in
a UAS-sc but not UAS-cul-2. (Zhang, Kusar, Vaessin; in revision).
31
tum is required for sensory organ precursors for suppression of sensory organ formation
Knock-down of tum with the pnr-GAL4 and en-GAL4 drivers (Figure 9) show animals
with a loss of bristles on the notum and missing wing regions, respectively. The wing phenotype
for the tum-RNAi line the en-GAL4 driver is different from that observed for cul-2-RNAi. In the
cul-2-RNAi fly, the wing is smaller and distorted. In the tum animal, part of the posterior
compartment is missing with a more jagged appearance. However at the imaginal wing disc
stage both cul-2-RNAi and tum-RNAi show ectopic A101 expression and SOP formation when
expressed with en-GAL4. tum is required for limited SOP development. In other words ectopic
SOP arise when tum expression levels are decreased or absent.
tum has a later function in development.
The temperature study of tum showed interesting results. There are two limitations in the
genetic tools being used to better understand the results. First, pnr-GAL4 and c765-GAL4 both
have different degrees of strength in driving expression of target constructs, with pnr-GAL4
apparently being stronger. Second, these animals have balancers, which when you add anything
that is not a wild type makes the animal sick to some degree. Furthermore, by stressing the
animals at a higher temperature to inactivate GAL80ts can further cause phenotypes that may be
contributed to what I will call the balancer effect. These limitations should be considered in the
interpretations of the results.
In comparison to wild type animals the temperature suppression using GAL-80ts showed
a later function of tum (Figure 11, 12). One sees two phenomena later in development from tum
suppression with the c765-GAL4 driver. First the “regular suppression” with GAL80ts at later
shifts show bigger wings compared to “unmodified expression”, no GAL80ts. This means tum
has an early function. However, “regular suppression” shows mild phenotypes of thicker bristles
32
in the wing marginal. This points to a later function of tum in addition to an early function. The
second observation to note is that the animals did show a phenotype that may be related to a
muscle attachment pathway because “regular suppression” flies showed defects in the
positioning of the wings in comparison to the “wild type”. Many of the wings pointed upwards in
the “regular suppression”. Conversely, “wild type” are laying flat. This could indicate a muscle
innovation. This will have to be explored later.
GAL80ts-׀pnr-GAL4>>UAS-tum-RNAi, on the other hand, showed different phenotypes.
One interesting new phenotype observed was that underneath the notum were what appeared to
be bulges in the epidermis of unknown origin in the “regular suppression” animals (Figure 11).
“Regular suppression” animals had loss of macro and microchaetae on the median notum
compared to “wild type”. “Unmodified expression” displayed loss of epidermis, and bristle
duplication.
tum has a function in bristle formation
Interesting bristle phenotypes appear throughout the study of cul-2 and tum. The adult
bristle is composed of four cells: the neuron, socket, shaft and sheath (Jarman, 2002). All four
cells derive two or three cell divisions from an SOP cell in the imaginal disc. With this in mind, a
possible explanation of the phenotypes could be the transformation of one cell into another
causing some of the phenotypes observed, such as three bristles (Figure 13), and double socket
and bristle (Figure 12). These types of phenotypes have been observed in several mutants that
function during later stages of sensory organ formation (Song and Lu 2012).
ModENCODE Temporal Expression Data from fly base further suggest that tum has a
later function. It appears that tum has a very high expression from 0-8 hours in development but
lower levels during first and second instar larva and moderate levels at third instar, and high
33
levels in adult females (Graveley et al., 2011). Also to note is the expression of tum in organ and
tissue is of moderate expression in CNS during larvae and ovary in adult (Chintapalli et al.,
2007).
In the mosaic analysis of tum, the FLP/FRT system previously discussed in this thesis
was utilized for mosaic/colonel analysis of tum. Colonel analysis was used to gain independent
insight in the specificity of tum gene function. The initial analysis showed animals with various
sensory organ phenotypes such as 3 shaft cells protruding at a single point. Further studies would
need to be done to trace the cells that make up a bristle to determine what cells are transforming
into this multiple shaft bristle. The results observed so far are in line with role of tum in SOP
and sensory organ formation.
tum in fred, tum in cul-2, and tum in sc interaction
Both tum and fred down regulated by the en-GAL4 driver in the same animal result in
animals that have the posterior part of the wing missing, but in a smooth appearance compared to
the single mutation of tum. This different phenotype was so far not universal among all animals,
to clearly state whether there are epistatic interactions between tum and fred,or if they act in
more of an additive observation. Further testing will need to be done to determine this.
The tum and cul-2 double mutation down regulated by the en-GAL4 driver showed much
variability. One explanation would be that each mutation by itself would have a level of
variability in the progeny. Often it is the animal with the weakest down regulation that survives.
However, when two mutations that have variability are combined the progeny may have the
potential to have even greater variability. This could help explain why some animals had small
wings, while other animals had even small wings. Others still had what looked like a gain of
wing veins. From this range of observation the same conclusion of the tum and fred mutation
34
resulted. There is inconclusive evidence at this point to argue how tum and cul-2 interact in
relations to one another in development.
The last double mutation genotype tested with en-GAL4 driver was tum-RNAi and sc-
RNAi. Achaete-scute complex (ASC) is a proneural gene in wing discs that provides SOP
potential. The complex is made of four basic Helix-Loop-Helix (bHLH) transcription factors one
which is scute (Vaessin et al., 1994). Previous studies of cul-2 and sc showed cul-2-associated
ectopic SOP requires the activity of the proneural acheatescute complex gene scute (Zhong,
Kusasr, Vaessin, in revision). If the results of tum and sc are anything like cul-2 and sc one
would suspect loss of ectopic SOP. However the results are mixed. Some wing disc show ectopic
SOP, while others do not. Further testing will need to be done to reach a more conclusive result.
35
Conclusion
Cul-2 and tum are required to prevent inappropriate sensory organ formation in
Drosophila. Both genes prevent epidermal cells from becoming neurogenic fates in the wing
disc. The cul-2 and tum losses of function phenotypes closely resemble the fred loss of function
phenotype. Tum has a later function in development. Future work will have to determine the
potential roles of cul-2 and tum in relation to fred function.
36
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